JCB: Report

How A destruction escapes the spindle assembly checkpoint

Barbara Di Fiore1,2 and Jonathon Pines1,2

1Wellcome Trust/Cancer Research UK and 2Department of Zoology, , Cambridge CB2 1QN, England, UK

he anaphase-promoting complex/cyclosome (APC/C) destruction, but how they enable checkpoint- is the ligase essential to , which en- resistant destruction has not been elucidated. In this study, T sures that specific proteins are degraded at spe- we answer this problem: we show that the N terminus of cific times to control the order of mitotic events. The APC/C cyclin A binds directly to Cdc20 and with sufficient affinity coactivator, Cdc20, is targeted by the spindle assembly that it can outcompete the SAC proteins. Subsequently, checkpoint (SAC) to restrict APC/C activity until meta- the Cks protein is necessary and sufficient to promote phase, yet early substrates, such as cyclin A, are degraded cyclin A degradation in the presence of an active check- in the presence of the active checkpoint. Cdc20 and the point by binding cyclin A–Cdc20 to the APC/C. cyclin-dependent kinase cofactor, Cks, are required for

Introduction Ubiquitin-mediated is fundamental to the proper underlie the degradation of cyclin A, which requires the Cks pro- control of mitosis. The anaphase-promoting complex/cyclosome tein to be degraded (Swan et al., 2005; Wolthuis et al., 2008). Cks (APC/C) ubiquitin ligase ensures the correct ordering of events proteins contain an anion-binding pocket implicated in binding by targeting specific proteins at specific times (Pines, 2006). to phosphoproteins (Bourne et al., 1996), including the APC/C The APC/C responds to the spindle assembly checkpoint (SAC) (Sudakin et al., 1997), whose phosphorylation by –Cdk such that it targets securin and cyclin B1 for destruction only at mitosis is crucial to its activity (Kraft et al., 2003). Because when all of the chromosomes have attached to the mitotic some cyclin A bound Cdc20 before mitosis, we proposed that its spindle, thereby ensuring that sister chromatids segregate to associated Cks subunit might direct the Cdc20–cyclin A complex opposite poles and that cells cannot exit mitosis until sister to the newly phosphorylated APC/C, thereby triggering cyclin A chromatids have separated. The SAC prevents the APC/C from destruction at mitotic entry (Wolthuis et al., 2008), but at that ubiquitylating securin and cyclin B1 by targeting its coactivator, time, we did not have any experimental proof for this model. Cdc20 (Musacchio and Salmon, 2007). However, Cdc20 is Our model also raised two important questions. First, if the THE JOURNAL OF CELL BIOLOGY not completely inactivated because it can still work with the Cks subunit does target Cdc20–cyclin A–Cdk to the APC/C, is this APC/C to ubiquitylate substrates such as cyclin A, Nek2A, and sufficient for destruction, or does the Cdk contribute? Cyclin A HOXC10 (den Elzen and Pines, 2001; Geley et al., 2001; Hames cannot be degraded in prometaphase if it can’t bind its Cdk or if et al., 2001; Gabellini et al., 2003; Wolthuis et al., 2008). How the Cdk can’t bind Cks (Stewart et al., 1994; den Elzen and APC/CCdc20 is able to recognize some substrates independently Pines, 2001; Geley et al., 2001; Wolthuis et al., 2008), but of the SAC but not others is crucial to our understanding of how whether the requirement to bind a Cdk is solely to recruit Cks is the APC/C and SAC cooperate to ensure genomic stability. not known. The second question is that because only a fraction One important insight is that the N terminus of Cdc20 is of cyclin A is bound to Cdc20 when cells enter mitosis, how is able to activate the APC/C in Xenopus laevis egg extracts (Kimata the rest of cyclin A degraded while the SAC is active? et al., 2008) to recognize the Nek2A protein that binds directly to The simplest hypothesis is that cyclin A can compete with the APC/C (Hayes et al., 2006). An analogous mechanism may the SAC proteins to bind Cdc20 and then be recruited to the APC/C via Cks. In this study, we provide strong support for this Correspondence to Jonathon Pines: [email protected] © 2010 Di Fiore and Pines This article is distributed under the terms of an Attribution– Abbreviations used in this paper: APC/C, anaphase-promoting complex/ Noncommercial–Share Alike–No Mirror Sites license for the first six months after the pub- cyclosome; DIC, differential interference contrast; FRT, flippase recognition tar- lication date (see http://www.rupress.org/terms). After six months it is available under a get; NEBD, nuclear envelope breakdown; SAC, spindle assembly checkpoint; Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, wt, wild type. as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

The Rockefeller University Press $30.00 J. Cell Biol. Vol. 190 No. 4 501–509 www.jcb.org/cgi/doi/10.1083/jcb.201001083 JCB 501 hypothesis by showing that cyclin A does not have to bind to degraded with normal kinetics in a Cdc20-dependent manner Cdc20 before mitosis to be degraded because the N terminus of (Fig. 2 A). Thus, cyclin A does not have to bind to Cdc20 before cyclin A binds directly to Cdc20 and can outcompete the SAC com- mitosis to be targeted for degradation in an SAC-resistant manner. plex for binding to Cdc20. Furthermore, we show that only Cks, and This supports our hypothesis that cyclin A binds to Cdc20 even not the Cdk subunit, is required for proteolysis because fusing the while the checkpoint is active. The first step in the SAC pathway N terminus of cyclin A to a Cks protein is sufficient to target it to is that Mad2 binds to Cdc20 (Musacchio and Salmon, 2007); the APC/C and confer destruction in an SAC-resistant manner. therefore, the simplest mechanism would be that cyclin A and Mad2 bind to overlapping regions of Cdc20. But we find that Results and discussion cyclin A binds to the WD40 repeat at the C terminus of Cdc20 (unpublished data), whereas the Mad2-binding region maps to Cdc20 binds directly to the N terminus the N terminus (aa 124–137; Yu, 2007). of cyclin A Because cyclin A and Mad2 did not compete for the same Previously, we showed that cyclin A bound to Cdc20 in G2 phase binding site on Cdc20, we tested the possibility that cyclin A and that both Cdc20 and Cks were required for its degradation could compete with the SAC complex for Cdc20 (note that by (Wolthuis et al., 2008). However, we did not determine the mecha- competition, we include mechanisms by which cyclin A could nism by which cyclin A could be degraded in an SAC-resistant preferentially bind Cdc20 by inducing an allosteric change). manner. To address this question, we first sought to identify the Because we find only a minor fraction of Mad2 bound to Cdc20 regions of cyclin A required to bind to Cdc20. (Nilsson et al., 2008), we tested whether cyclin A could displace An extended sequence in the N terminus of cyclin A is BubR1. We immunoprecipitated Cdc20 from nocodazole-treated required to target cyclin A for degradation in prometaphase cells, added purified GST fusion proteins, and assayed their (den Elzen and Pines, 2001; Geley et al., 2001; Jacobs et al., 2001). effect on the integrity of the Cdc20–SAC complex. Consistent We generated several cyclin A deletion mutants (Fig. 1 A) tagged with our competition hypothesis, excess cyclin A displaced at the C terminus with the Flag epitope and a fluorescent protein BubR1 from Cdc20 (Fig. 2 B), and this depended on its ability to (Venus; Nagai et al., 2002). As a control, we used cyclin B1, interact with Cdc20; full-length cyclin A and the 1–165 fragment which is degraded in an SAC-sensitive manner (Clute and Pines, were both able to displace BubR1, whereas neither the 1–98 frag- 1999). To ensure that the proteins were expressed at close to en- ment of cyclin A nor the N terminus of cyclin B1 containing its D dogenous levels, we generated stable inducible HeLa cell lines box (1–167) could do so (Fig. 2, C and D). We added a large ex- in which a single copy of the plasmid was integrated into the ge- cess (10–50-fold over Cdc20-coimmunoprecipitated BubR1) of nome at a flippase recognition target (FRT) site. The proteins the proteins in these in vitro reactions, but it is reasonable to sup- were immunoprecipitated from prometaphase cells using an pose that the in vitro conditions do not recapitulate those in vivo anti-Flag antibody and probed for Cdc20 and Cdk2. As expected, and that competition in vivo may be more efficient. the N-terminal fragments lacking the cyclin box could not bind To test whether cyclin A could compete with BubR1 for Cdk2 (Fig. 1, B and C). Cdc20 in vivo, we overexpressed cyclin A or cyclin A 1–165 Consistent with our previous findings (Wolthuis et al., 4–12-fold over endogenous and found that both greatly reduced 2008), cyclin A, but not cyclin B1, strongly bound to Cdc20, but the level of Cdc20 in BubR1 immunoprecipitates (Fig. 2, E and F). neither the N-terminal fragment (1–98) containing the extended Thus, we conclude that both in vitro and in vivo, cyclin A is able motif required for cyclin A degradation nor the complementary to compete with the SAC proteins for binding to Cdc20, even mutant N97 were able to bind Cdc20 (Fig. 1, B and C), indicat- when Cdc20 is already bound to the SAC complex. ing that a more complex region was required. A longer N-terminal fragment, 1–165, did bind Cdc20 to a similar extent as full-length Binding to Cdc20 is necessary but not cyclin A (Fig. 1, B and C), which is in agreement with results from sufficient for the proper timing of a yeast two-hybrid analysis (Ohtoshi et al., 2000). Moreover, cyclin A degradation cyclin A and Cdc20 bound directly to each other (Fig. 1 D). Having identified a region of cyclin A sufficient to bind to Cdc20, Complementing these data, an internal deletion that re- regardless of whether Cdc20 was free or bound in an SAC com- moved the extended D-box motif (47–83) abrogated Cdc20 plex, we asked whether this was sufficient to target a protein for binding (Fig. 1, B and C) when expressed at similar levels to en- destruction in prometaphase. We used Venus-tagged proteins to dogenous cyclin A. We conclude that the extended D-box motif assay the precise timing of destruction in mitosis and compared contributes to Cdc20 binding, but is not itself sufficient, and that full-length cyclin A, which is degraded as soon as cells begin another region between residues 98 and 165 is required. prometaphase (Fig. 3 A; den Elzen and Pines, 2001; Geley et al., 2001; Wolthuis et al., 2008), with both the N-terminal fragments Cyclin A competes with BubR1 for and a chimeric protein replacing the N terminus of cyclin B1 Cdc20 binding with that of cyclin A. We previously showed that some, but by no means all, cyclin A In support of our hypothesis, the N terminus of cyclin A binds to Cdc20 in G2 phase (Wolthuis et al., 2008), raising the conferred the ability to be degraded in an SAC-resistant man- question of what happened to cyclin A that was not bound to ner on cyclin B1 (Fig. 3 B), whereas the cyclin A 1–98 frag- Cdc20. To answer this, we injected purified cyclin A–GFP–Cdk2 ment that could not bind Cdc20 was not degraded until anaphase lacking Cdc20 into prometaphase cells and found that this was (Fig. 3, C and D; note that data are plotted twice, once normalized

502 JCB • VOLUME 190 • NUMBER 4 • 2010 Figure 1. The N terminus of cyclin A binds Cdc20. (A) Schematic representation of cyclin A mutants. (B) Stable inducible HeLa FRT cell lines expressing cyclin B1, and the cyclin A mutants were synchronized in mitosis by a single thymidine block and released in the presence of nocodazole. To prevent cyclin A degradation, MG132 was added 2 h before collecting mitotic cells by shake off. Cells were lysed, and anti-Flag immunoprecipitates (IP) were probed for Cdc20, Cdk2, and Flag. (C) Cdc20 and Cdk2 levels in the anti-Flag immunoprecipitates (B) were quantified using an Odyssey scanner, corrected for the level of Flag-tagged protein, and normalized to the amount bound to wt cyclin A. Error bars indicate mean ± SEM from three experiments. (D) GST–cyclin A was purified from bacteria and incubated for 2 h at 4°C with His6-Cdc20 purified from insect cells. Cyclin A was isolated on glutathione beads, and the beads and supernatants (SPN) analyzed by immunoblots were probed for Cdc20 and GST. Values are representative of seven experiments. Molecular mass markers are shown on the left (kilodaltons). to nuclear envelope breakdown [NEBD] and once to anaphase). SAC is satisfied. Therefore, binding directly to Cdc20 is not in In contrast, the 1–165 fragment that bound Cdc20 was degraded itself sufficient to confer SAC-resistant proteolysis. Thus, addi- earlier, but notably, this was in metaphase not prometaphase (Fig. 3, tional factors must contribute to the SAC-resistant degradation E and F). Because metaphase equates to when the SAC has been of cyclin A. satisfied, we tested whether this destruction was under the control of the SAC. We depleted Mad2 to inactivate the SAC and found Cks1 is essential to mediate checkpoint- this advanced cyclin A (1–165) degradation to begin just after resistant degradation NEBD (Fig. 3, I and J), which is consistent with regulation by To identify the missing component that confers SAC-resistant the SAC. In contrast, degradation of the 1–98 fragment was still destruction, we focused first on the Cks protein because full- delayed until anaphase (Fig. 3, G and H). length cyclin A must bind to its Cdk partner and consequently We conclude that although the 1–165 fragment can com- to Cks to be degraded (Wolthuis et al., 2008). Moreover, the pete with BubR1 for Cdc20, it can only be degraded when the N terminus of cyclin A was sufficient to confer SAC-resistant

How cyclin A destruction escapes the checkpoint • Di Fiore and Pines 503 Figure 2. Cyclin A competes with BubR1 for binding to Cdc20. (A) The cyclin A–GFP–Cdk2 complex was purified from baculovirus-infected insect cells and injected into prometaphase HeLa cells identified by DIC (top). Time is rela- tive to time of injection (min). Bar, 10 µm. GFP fluorescence through mitosis was measured (bottom) for normal (black curve) and Cdc20- depleted (gray curve) cells and set to 100 at the time of injection. Error bars indicate mean ± SD of 22 cells from two experiments (control) and 26 cells from two experiments (siCdc20). (B) Cdc20 bound in the SAC complex was immunoprecipitated (IP) with anti-Cdc20 anti- bodies from nocodazole-arrested cells and incubated with GST alone or GST–cyclin A for 2 h at 4°C. The supernatant (SPN) was removed and analyzed by immunoblotting for BubR1, Cdc20, and GST. Black line indicates that intervening lanes have been spliced out. (C) Cdc20 immunoprecipitates (obtained as in B) were incubated with buffer (mock), GST, the indicated GST–cyclin A mutants, or GST– cyclin B1 (1–167) and analyzed as in B. (D) The amount of BubR1 released from the immunocomplex (C) was quantified using an Odyssey scanner, corrected for the amount of Cdc20 immunoprecipitated and normalized to that released by wt cyclin A. Error bars indicate mean ± SEM from at least five experiments. (E) Stable cell lines expressing either Venus alone, cyclin A–Venus, or cyclin A (1–165)– Venus were treated with nocodazole, and MG132 was added 2 h before lysis. Anti- BubR1 immunoprecipitates were probed for BubR1, Cdc20, and Venus. (F) The amount of Cdc20 associated with immunoprecipitated BubR1 (E) was quantified as in D and- cor rected for the amount of immunoprecipitated BubR1 (light gray). To compare the wt and 1–165 samples, the values were corrected for expression levels, setting the 1–165-Venus level to 1 (dark gray). Error bars indicate mean ± SEM from four experiments. (B, C, and E) Input represents 1/10 of the immunoprecipitation. Molecular mass markers are shown on the left (kilodaltons).

degradation on cyclin B1 (Fig. 3 B) that binds Cks via its Cdk1 anaphase (Fig. 4, A and B). Similarly, fusing Cks1 to the N terminus partner. However, it was unclear whether the Cdk subunit itself of cyclin B1 that cannot bind Cdc20 when the SAC is active did not contributed to the destruction of cyclin A. change the timing of its destruction to prometaphase, although it Because we hypothesized that the role of Cks was to bind made it a more efficient substrate (unpublished data; seevan Zon the APC/C, we predicted that we could bypass the Cdk and target et al. in this issue). the cyclin A 1–165 fragment for degradation even in the presence These results demonstrate that Cks1 is sufficient to of an active SAC by fusing it to a Cks protein. In confirmation of confer SAC-resistant degradation on a cyclin A–Cdc20 com- this, a cyclin A (1–165)–Venus-Cks1 fusion protein was degraded plex, bypassing any requirement for Cdk binding or activity just after NEBD (Fig. 4, C and D) with the same timing as wild- (Fig. S1, A and B). Thus, the primary reason why cyclin A has type (wt) cyclin A. Furthermore, fusing Cks1 to cyclin A (1–98) to bind to its Cdk to be degraded is to be targeted to the APC/C that could not bind Cdc20 had no effect; it was still degraded in via the Cks protein.

504 JCB • VOLUME 190 • NUMBER 4 • 2010 Figure 3. Binding to Cdc20 only confers SAC- dependent destruction. (A) HeLa cells were in- jected in G2 phase with cyclin A–Venus-Flag, and the fluorescence was measured through mitosis. Fluorescence at NEBD is set to 100. Error bars indicate mean ± SD of 36 cells. Time is relative to NEBD. (B) Mean degradation curve of a chimera between the N terminus of cyclin A (1–165) and C terminus of cyclin B1 (171–433) obtained as in A in the absence (black curve) or presence (gray curve) of nocodazole. Error bars indicate mean ± SD of 49 cells from three experiments (untreated) and 27 cells from two experiments (nocodazole). (C–F) Mean deg- radation curve of cyclin A (1–98)–Venus-Flag (C and D) and cyclin A (1–165)–Venus-Flag (E and F) obtained as in A. Time is relative to NEBD (C and E) or anaphase (D and F), and fluorescence at NEBD or anaphase is set to 100. Error bars indicate mean ± SD of 12 cells from three experiments (C and D) and 36 cells from three experiments (E and F). (G–J) Mean deg- radation curve of cyclin A (1–98)–Venus-Flag (G and H) and cyclin A (1–165)–Venus-Flag (I and J) in Mad2-depleted cells were obtained as in A except that the Mad2 siRNA oligonucleo­ tides were transfected during synchronization. Because inactivating the SAC accelerates pro- gression from NEBD to anaphase (not depicted; Meraldi et al., 2004), we only considered those cells in which NEBD to anaphase was shortened from the 40 min in control cells to <20 min. Time is relative to NEBD (G and I) or anaphase (H and J). Error bars indicate mean ± SD of 10 cells (G and H) and 13 cells from three experi- ments (I and J).

Fusion to Cks1 promotes proteins (Fig. 4 E, lanes 10–19). Cks increased the processivity cyclin A ubiquitylation of cyclin A ubiquitylation because although the amount of If Cks does target cyclin A to the APC/C, one might predict mono- and oligoubiquitin species (one to five ) was that it should enhance cyclin A ubiquitylation. Therefore, we similar for the two substrates (Fig. 4 F), the proportion of poly- compared the in vitro ubiquitylation of cyclin A (1–165)– ubiquitin chains was higher for the Cks1 fusion (Fig. 4 G). More- Venus (Fig. 4 E, lanes 1–9) and cyclin A (1–165)–Venus-Cks1 over, cyclin A (1–165)–Cks1 was clearly a better competitor in

How cyclin A destruction escapes the checkpoint • Di Fiore and Pines 505 Figure 4. Cyclin A–associated Cks1 increases the efficiency of cyclin A ubiquitylation. (A–D) Cells were injected in G2 phase with cyclin A (1–98)–Venus-Cks1 (A and B) or cyclin A (1–165)–Venus-Cks1 (C and D), and the fluorescence was measured through mitosis. Time is relative to NEBD (A and C) or anaphase (B and D). Error bars indicate mean ± SD of 19 cells from two experiments (A and B) or 24 cells from two experiments (C and D). (E) In vitro ubiquitylation assay of cyclin A (1–165)–Venus (lanes 1–9) or cyclin A (1–165)–Venus-Cks1 (lanes 10–19). Reactions were performed for the indicated time (shown in minutes) before analysis by SDS-PAGE and phosphoimaging. Control reactions are without APC/C (lanes 9 and 18) or E2 (lane 19). (F and G) Quantification of ubiquitin conjugates in E with one to five ubiquitin (F) and more than five ubiquitin (G) molecules were normalized to the total amount of ubiquitylated substrate. (F and G) Error bars indicate mean ± SEM from four experiments. (H) Ubiquitylation reactions for cyclin A (1–165)–Venus (lanes 1–4) and cyclin A (1–165)– Venus-Cks1 (lanes 5–8). 100 times excess of unlabeled cyclin A (1–165)–Venus (lanes 3 and 7) or cyclin A (1–165)–Venus-Cks1 (lanes 4 and 8) was added as a competitor at the beginning of the reaction. (I) Quantification of reactions in H. Numbers correspond to lanes in H. *, unmodified substrate; **, P < 0.01; ***, P < 0.05 (calculated using Student’s t test).

506 JCB • VOLUME 190 • NUMBER 4 • 2010 Figure 5. Cks directly recruits cyclin A to the APC/C. (A) Purified His6-Cks1 and an anion-binding site mutant (R20A) and fusion proteins between cyclin A (1–165)–Venus and wt or (R20A) mutant Cks1 were immobilized on beads and incubated with extract from nocodazole-treated HeLa cells. Beads were analyzed by SDS-PAGE and immunoblotted for APC3, cyclin A, and Cks1. Molecular mass markers are shown on the left (kilodaltons). (B) APC3 binding to Cks1 (A) was quantified on an Odyssey scanner and corrected for the amount of Cks1. Values were normalized to wt Cks1. Error bars indicate mean ± SEM from three experiments. (C and D) Degradation of cyclin A (1–165)–Venus-Cks1 (R20A) measured as in Fig. 4 (A and B). Time is relative to NEBD (C) or anaphase (D). Error bars indicate mean ± SD of 28 cells from two experiments. (E) Working model. The cyclin A–Cdk–Cks complex binds to soluble or APC/C-associated Cdc20 by out competing the SAC complex. Recruitment to the APC/C is mediated by Cks interaction with phosphorylated APC/C. in vitro APC/C-dependent ubiquitylation reactions than cyclin A binding to a Cdk was not affected (Fig. 5, A and B; and not (1–165; Fig. 4, H and I). depicted). When fused to cyclin A (1–165), Cks1 (R20A) was To test whether the Cks protein had to bind to the APC/C unable to confer SAC-resistant degradation; like the 1–165 to promote cyclin A degradation in vivo, we mutated the anion- fragment alone, the R20A fusion protein could only be degraded binding site of Cks1 (R20A; Watson et al., 1996). This mutant in metaphase once the SAC was satisfied (Fig. 5, C and D). was severely disabled in its ability to bind to the APC/C, whereas Thus, the anion-binding site of Cks1 was essential to confer

How cyclin A destruction escapes the checkpoint • Di Fiore and Pines 507 SAC-resistant degradation on a cyclin A–Cdc20 complex. This Fusions between cyclin A fragments and Cks1 were constructed in modi- result strongly indicates that Cks1 mediates the recruitment of fied versions of the pEYFP-N1 plasmid, resulting in the YFP/Venus sequence expressed between cyclin A and Cks1. For the cyclin A-B1 fusion protein, cyclin A to a phosphorylated APC/C subunit. Several APC/C cyclin A N terminus (aa 1–165) was fused to the C terminus of cyclin B1 subunits are phosphorylated during mitosis (Kraft et al., 2003; (aa 171–433) in a modified version of pEYFP-N1 plasmid. For protein Steen et al., 2008), of which APC3 is most likely to bind Cks. purification, pGEX (GE Healthcare), a modified version of pET30a (EMD) containing a strep tag, and pRSET (Invitrogen) vectors were used. We conclude that two main factors contribute to the SAC- resistant degradation of cyclin A. First, cyclin A can bind directly to Microinjection and time-lapse imaging and analysis Cdc20 with sufficient affinity to displace SAC proteins. Second, the For microinjection and time-lapse microscopy, the culture medium was re- placed with Leibovitz’s L-15 medium (Invitrogen). Plasmids or recombinant Cks protein targets a cyclin A–Cdk complex to the APC/C in a man- protein were microinjected into G2 or prometaphase cells, respectively, ner that allows Cdc20 bound to cyclin A to activate the APC/C. using a semiautomatic microinjector (Eppendorf) on a microscope (DMIRBE Currently, we do not know exactly how cyclin A competes or DMIRE2; Leica). Differential interference contrast (DIC) and fluorescence images were captured every 3 min with a charge-coupled device camera with the SAC proteins for Cdc20, in part because there is some (QuantEM 512B; Photometrics) using Slidebook software (Intelligent Imag- disagreement over how the SAC proteins themselves bind Cdc20. ing Innovations). A modified version of ImageJ software (National Institutes We and others find that the majority of Cdc20 binds to an SAC of Health) was used to quantify the fluorescence after background subtrac- tion. DIC microscopy was used to monitor mitotic phases. complex with BubR1 and Bub3 but only a small fraction of Mad2, whereas others find that Mad2 forms a stoichiometric part of RNA interference the complex (the reason for this discrepancy is unclear but siRNA duplex against Mad2 (5-GGAAGAGUCGGGACCACAGUU-3; Thermo Fisher Scientific), Cdc20 (5-CGGAAGACCTGCCGTTACA-3; may be attributable to the methods used to lyse the cells and iso- Thermo Fisher Scientific), and control siRNA duplex against GAPDH late the SAC complexes or the methods used to calculate the (Applied Biosystems) were transfected at a final concentration of 100 nM amounts of protein in the complex; Nilsson et al., 2008; Herzog with Oligofectamine (Invitrogen) according to the manufacturer’s protocol. Transfection was performed during synchronization 5 h after release from et al., 2009; Kulukian et al., 2009). However, there is general the thymidine block. For Cdc20, an extra transfection was performed be- agreement that Mad2 is required for Cdc20 to bind to BubR1 fore starting the synchronization protocol. in vivo; therefore, if cyclin A blocks access to the Mad2-binding Protein expression and pull-downs site on Cdc20, either sterically or by inducing a conformational Recombinant proteins were induced with 0.5 mM IPTG in BL21 (DE3) at change, this would prevent BubR1 from binding. Alternatively, 25°C for 5 h and purified on glutathione Sepharose 4B beads (GE Health- as suggested by our in vitro assays, cyclin A and BubR1 them- care), Strep-Tactin matrix (IBA), or nickel beads (QIAGEN) according to the manufacturer’s protocols. His-tagged Cdc20 was purified from baculovirus- selves might compete for Cdc20. infected Sf9 cells as previously described (Nilsson et al., 2008). For GST Because both cyclin A and cyclin B1 require Cdc20 for pull-downs, GST proteins were immobilized on glutathione beads and in- their degradation, is the difference between them simply that cubated with purified Cdc20 in buffer A (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM MgCl , 5% glycerol, and 1 mM DTT) with 10 µg BSA for 2 h cyclin A has a greater affinity for Cdc20, or might it bind to Cdc20 2 at 4°C. For His6-Cks1 and cyclin A–Cks-strep–binding assays, purified pro- in a different manner that allows SAC-independent destruction? teins were coupled to nickel or Strep-Tactin beads and incubated in buffer We think the latter idea is unlikely because the SAC inhibits de- (50 mM Tris-HCl, pH 7.8, 150 mM NaCl, and 0.2% NP-40) for 2 h at 4°C with extracts from nocodazole-treated HeLa cells. For protein microinjection, struction of cyclin A (1–165) that binds Cdc20. Thus, our inter- cyclin A–GFP–His–Cdk2 complex was purified from baculovirus-infected pretation is that cyclin A can be degraded by either or both of two insect cells using nickel beads, and the folding of the purified complex was pathways (Fig. 5 E). Cyclin A can either compete for Cdc20 be- assessed on a gel filtration column (Superdex 200; GE Healthcare). fore being targeted to the APC/C by Cks, or a cyclin A–Cdk–Cks Immunoprecipitation and competition experiments complex could bind to an APC/C to which Cdc20 is already Cells were lysed in 50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 0.5% bound as part of an SAC complex, and, once bound, the N termi- NP-40 plus protease inhibitor cocktail (Roche), and 10 nM microcystin (LGC- Promotech) for 20 min on ice and clarified by a 12,000 g spin for 15 min nus of cyclin A could displace the SAC proteins from Cdc20, at 4°C. Complexes were immunoprecipitated for 2 h at 4°C with anti-Flag thereby activating the APC/C. (M2; Sigma-Aldrich) or anti-BubR1 (BD) antibody covalently coupled to pro- tein G (Dynabeads; Invitrogen). After five washes in lysis buffer, proteins were eluted from beads by incubating for 5 min at 65°C in sample buffer. For the competition experiments, Cdc20 complexes were isolated using anti-Cdc20 Materials and methods (H-7; Santa Cruz Biotechnology, Inc.) antibody. After the washes, beads with associated complexes were divided into several tubes, and 1–2 µM purified Cell culture and synchronization GST fusion protein was added to the beads and incubated in buffer A for HeLa cells were cultured in advanced DME (Invitrogen) supplemented with 2% 2 h at 4°C. The supernatant containing the unbound GST proteins and pro- fetal bovine serum. Cells were synchronized by a thymidine/aphidicolin pro- teins released from the immunoprecipitated complexes were analyzed by tocol as previously described (Di Fiore and Pines, 2007). Cells were blocked immunoblotting. Beads were washed three times with buffer A, and the com- with 2.5 mM thymidine (Sigma-Aldrich) for 24 h, released for 12 h, and plexes still associated eluted in sample buffer for 5 min at 65°C. blocked again with 5 µg/ml aphidicolin (Sigma-Aldrich) for 24 h. Cells were released into fresh medium. For prometaphase arrest, 0.1 µg/ml nocodazole Immunoblotting (Sigma-Aldrich) or 100 nM taxol (Sigma-Aldrich) was added during release After electrophoresis on 4–12% gradient Bis-Tris acrylamide gels, proteins from a single thymidine block, and 12 h later, cells were either harvested by were transferred to a PVDF membrane. Membrane saturation and all of the mitotic shake off or 10 µM MG132 (EMD) was added, and cells collected following incubation steps were performed in 5% low fat milk in 0.1% PBS- by mitotic shake off 2 h later. The HeLa-FRT cell lines (provided by S. Taylor, Tween. Anti-Flag (M2; Sigma-Aldrich), anti-GST (B-14; Santa Cruz Biotechnol- University of Manchester, Manchester, England, UK) were transfected using ogy, Inc.), anti–cyclin A (Cancer Research UK), and anti-APC3 (BD) were the FLIP-in system (Invitrogen) to generate stable inducible cell lines. Cells were used at 1:1,000. Anti-Cdc20 (Bethyl Laboratories), anti-BubR1 (Bethyl Labo- induced with 1 µg/ml tetracycline (EMD) 12 h before harvesting. ratories and BD), and anti-Cdk2 (Koop, 2007) were used at 1:500. Anti- Cks1 (Invitrogen) was used at 1:150. Alexa Fluor 680 (Invitrogen)– and Plasmids IRDye (800CW; LI-COR Biosources)-conjugated secondary antibodies were Cyclin A and its mutants were cloned into a modified version of pCDNA5/ used at 1:5,000. The antibody signal was detected using the infrared imag- FRT/TO (Invitrogen) containing Venus-Flag tag using the Gateway system. ing system (Odyssey; LI-COR) for quantitative immunoblotting.

508 JCB • VOLUME 190 • NUMBER 4 • 2010 APC/C purification and ubiquitylation assays Jacobs, H.W., E. Keidel, and C.F. Lehner. 2001. A complex degradation signal in Taxol-treated HeLa cells were resuspended in 20 mM Hepes, pH 7.8, 175 mM cyclin A required for G1 arrest, and a C-terminal region for mitosis. EMBO J. 20:2376–2386. doi:10.1093/emboj/20.10.2376 NaCl, 2.5 mM MgCl2, 10% glycerol, 1 mM DTT, 1 mM PMSF, protease inhibitor cocktail (Roche), 2 µM ocadaic acid, 10 nM microcystin LR, and Kimata, Y., J.E. Baxter, A.M. Fry, and H. Yamano. 2008. A role for the Fizzy/ phosphatase inhibitor cocktail II (EMD) and ruptured using nitrogen cavita- Cdc20 family of proteins in activation of the APC/C distinct from substrate Mol. Cell. tion. APC/C was immunoprecipitated from 10 mg of extract using anti-APC4 recruitment. 32:576–583. doi:10.1016/j.molcel.2008.09.023 antibodies immobilized on protein G (Dynabeads; Invitrogen). Substrates Koop, L. 2007. The role of Cyclin A in the entry to mitosis. PhD thesis. University were purified on Strep-Tactin beads (IBA) and radiolabeled with33 P by phos- of Cambridge, Cambridge, England, UK. 176 pp. phorylation of a muscle kinase tag (HMK) using protein kinase A (Sigma- Kraft, C., F. Herzog, C. Gieffers, K. Mechtler, A. Hagting, J. Pines, and J.M. Peters. Aldrich). Ubiquitylation reactions were performed at 37°C in 15 µl of QPIP 2003. Mitotic regulation of the human anaphase-promoting complex by phosphorylation. EMBO J. 22:6598–6609. doi:10.1093/emboj/cdg627 buffer (50 mM Pipes, pH 7.5, 100 mM NaCl, 2 mM MgCl2, 10% glycerol, 1 mM DTT, and 1 mM EGTA) containing 5 µl APC4 beads, 300 nM E1, Kulukian, A., J.S. Han, and D.W. Cleveland. 2009. Unattached kinetochores 2.6 µM UbcH10, 150 µM ubiquitin, 1 µM BSA, 100 nM CDC20, 100 nM catalyze production of an anaphase inhibitor that requires a Mad2 tem- radiolabeled substrate, 2 mM ATP, 2.3 µM creatine kinase, and 10 mM cre- plate to prime Cdc20 for BubR1 binding. Dev. Cell. 16:105–117. doi:10 .1016/j.devcel.2008.11.005 atine phosphate as previously described (Garnett et al., 2009). For the com- petition with cold (unlabeled) substrate, a 100 times excess over the labeled Meraldi, P., V.M. Draviam, and P.K. Sorger. 2004. Timing and checkpoints in the substrate was added to each reaction at time 0. Reactions were stopped with regulation of mitotic progression. Dev. Cell. 7:45–60. doi:10.1016/ j.devcel.2004.06.006 SDS sample buffer, processed for SDS-PAGE, and analyzed on a phospho­ imager (FLA-5000; Fujifilm). Musacchio, A., and E.D. Salmon. 2007. The spindle-assembly checkpoint in space and time. Nat. Rev. Mol. Cell Biol. 8:379–393. doi:10.1038/nrm2163 Nagai, T., K. Ibata, E.S. Park, M. Kubota, K. Mikoshiba, and A. Miyawaki. Online supplemental material 2002. A variant of yellow fluorescent protein with fast and efficient Fig. S1 shows the strong reduction in Cdk2 binding of the cyclin A (1–165)– maturation for cell-biological applications. Nat. Biotechnol. 20:87–90. Venus-Cks1 protein compared with full-length cyclin A. Online supple- doi:10.1038/nbt0102-87 mental material is available at http://www.jcb.org/cgi/content/full/jcb Nilsson, J., M. Yekezare, J. Minshull, and J. Pines. 2008. The APC/C maintains .201001083/DC1. the spindle assembly checkpoint by targeting Cdc20 for destruction. Nat. Cell Biol. 10:1411–1420. doi:10.1038/ncb1799 We thank Steven Taylor for the gift of the parental HeLa-FRT cell line. We are also Ohtoshi, A., T. Maeda, H. Higashi, S. Ashizawa, and M. Hatakeyama. 2000. grateful to all members of our laboratory for stimulating discussions and to Claire Human p55(CDC)/Cdc20 associates with cyclin A and is phosphorylated Acquaviva, who contributed to the early stage of the project. by the cyclin A-Cdk2 complex. Biochem. Biophys. Res. Commun. 268: This work was supported by a program grant from Cancer Research UK 530–534. doi:10.1006/bbrc.2000.2167 (to J. Pines) and by a core grant (to the Gurdon Institute) from Cancer Research UK Pines, J. 2006. Mitosis: a matter of getting rid of the right protein at the right and The Wellcome Trust. time. Trends Cell Biol. 16:55–63. doi:10.1016/j.tcb.2005.11.006 Steen, J.A., H. Steen, A. Georgi, K. Parker, M. Springer, M. Kirchner, F. Submitted: 18 January 2010 Hamprecht, and M.W. Kirschner. 2008. Different phosphorylation states Accepted: 20 July 2010 of the anaphase promoting complex in response to antimitotic drugs: a quantitative proteomic analysis. Proc. Natl. Acad. Sci. USA. 105:6069– 6074. doi:10.1073/pnas.0709807104 Stewart, E., H. Kobayashi, D. Harrison, and T. Hunt. 1994. Destruction of References Xenopus A and B2, but not B1, requires binding to p34cdc2. EMBO J. 13:584–594. Bourne, Y., M.H. Watson, M.J. Hickey, W. Holmes, W. Rocque, S.I. Reed, and J.A. Tainer. 1996. Crystal structure and mutational analysis of the human Sudakin, V., M. Shteinberg, D. Ganoth, J. Hershko, and A. Hershko. 1997. CDK2 kinase complex with -regulatory protein CksHs1. Cell. Binding of activated cyclosome to p13(suc1). Use for affinity purifica- 84:863–874. doi:10.1016/S0092-8674(00)81065-X tion. J. Biol. Chem. 272:18051–18059. doi:10.1074/jbc.272.29.18051 Clute, P., and J. Pines. 1999. Temporal and spatial control of cyclin B1 destruc- Swan, A., G. Barcelo, and T. Schüpbach. 2005. Drosophila Cks30A interacts tion in metaphase. Nat. Cell Biol. 1:82–87. doi:10.1038/10049 with Cdk1 to target cyclin A for destruction in the female germline. Development. 132:3669–3678. doi:10.1242/dev.01940 den Elzen, N., and J. Pines. 2001. Cyclin A is destroyed in prometaphase and can delay chromosome alignment and anaphase. J. Cell Biol. 153:121–136. van Zon, W., J. Ogink, B. ter Riet, R.H. Medema, H. te Riele, and R.M.F. doi:10.1083/jcb.153.1.121 Wolthuis. 2010. The APC/C recruits cyclin B1–Cdk1–Cks in prometa- phase before D box recognition to control mitotic exit. J. Cell Biol. Di Fiore, B., and J. Pines. 2007. Emi1 is needed to couple DNA replication with 190:587–602. doi:10.1083/jcb.200912084 mitosis but does not regulate activation of the mitotic APC/C. J. Cell Biol. 177:425–437. doi:10.1083/jcb.200611166 Watson, M.H., Y. Bourne, A.S. Arvai, M.J. Hickey, A. Santiago, S.L. Bernstein, J.A. Tainer, and S.I. Reed. 1996. A mutation in the human cyclin-dependent Gabellini, D., I.N. Colaluca, H.C. Vodermaier, G. Biamonti, M. Giacca, A. kinase interacting protein, CksHs2, interferes with cyclin-dependent kinase Falaschi, S. Riva, and F.A. Peverali. 2003. Early mitotic degradation of binding and biological function, but preserves protein structure and assembly. the homeoprotein HOXC10 is potentially linked to cell cycle progression. J. Mol. Biol. 261:646–657. doi:10.1006/jmbi.1996.0490 EMBO J. 22:3715–3724. doi:10.1093/emboj/cdg340 Wolthuis, R., L. Clay-Farrace, W. van Zon, M. Yekezare, L. Koop, J. Ogink, R. Garnett, M.J., J. Mansfeld, C. Godwin, T. Matsusaka, J. Wu, P. Russell, J. Pines, Medema, and J. Pines. 2008. Cdc20 and Cks direct the spindle check- and A.R. Venkitaraman. 2009. UBE2S elongates ubiquitin chains on point-independent destruction of cyclin A. Mol. Cell. 30:290–302. doi: APC/C substrates to promote mitotic exit. Nat. Cell Biol. 11:1363–1369. 10.1016/j.molcel.2008.02.027 doi:10.1038/ncb1983 Yu, H. 2007. Cdc20: a WD40 activator for a cell cycle degradation machine. Geley, S., E. Kramer, C. Gieffers, J. Gannon, J.-M. Peters, and T. Hunt. 2001. Mol. Cell. 27:3–16. doi:10.1016/j.molcel.2007.06.009 Anaphase-promoting complex/cyclosome–dependent proteolysis of human cyclin A starts at the beginning of mitosis and is not subject to the spindle assembly checkpoint. J. Cell Biol. 153:137–148. doi:10.1083/jcb .153.1.137 Hames, R.S., S.L. Wattam, H. Yamano, R. Bacchieri, and A.M. Fry. 2001. APC/C- mediated destruction of the centrosomal kinase Nek2A occurs in early mitosis and depends upon a cyclin A-type D-box. EMBO J. 20:7117– 7127. doi:10.1093/emboj/20.24.7117 Hayes, M.J., Y. Kimata, S.L. Wattam, C. Lindon, G. Mao, H. Yamano, and A.M. Fry. 2006. Early mitotic degradation of Nek2A depends on Cdc20- independent interaction with the APC/C. Nat. Cell Biol. 8:607–614. doi: 10.1038/ncb1410 Herzog, F., I. Primorac, P. Dube, P. Lenart, B. Sander, K. Mechtler, H. Stark, and J.-M. Peters. 2009. Structure of the anaphase-promoting complex/cyclo- some interacting with a mitotic checkpoint complex. Science. 323:1477– 1481. doi:10.1126/science.1163300

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